hi,
I am working on a membrane protein of 153 kDa mass. I eluted it with 300mM imidazole by Ni NTA. Does the centrifugal filter (100kDa cutoff) which I am using to concentrate my protein will remove the imidazole (as far as I know imidazole is around 68kDa)?
Hi,
The imidazole will go through the filter, as its molar mass is 68 Da (not kDa). However, in order to remove it, you will need to top the protein fraction up with buffer without imidazole and concentrate again.
For example, if you started with 10 mL 300 mM imidazole and concentrated to 1 mL, then added 9 mL of buffer with no imidazole and concentrated again to 1 mL, you would have 10 x concentrated protein in 30 mM imidazole. Do the same again and you would have 3 mM imidazole, etc.
Hi there, If u have to go that way only, then I recommend previous answer by D Crabtree. Or I suggest you to dialyzes the eluted protein against the dialysis buffer not having Imidazole in it, for 6-8 hr at 4C with magnetic stirrer on. This way most of the imidazole will be out of the solution prior to you concentrate it.
Hope it helps!
Hi there,
A other alternative to get rid of imidazole is to pass your sample on a desalting column (PD-10 for instance) using your imidazole free buffer as the eluant. Then because desalting tend to dilute your protein of interest, concentration on vivaspin is recommended. The desalting step may also be performed in a more accurate fashion by running SEC on appropriate superdex or superose column. The main advantage of this step is that you will get information on the homogeneity/heterogeneity of your protein by looking at the elution profile (single peak or multiple peaks).
Concentrating the protein with sequential dilution (on column) is supposed to take long time (hours), If you have tried it, have you observed instability in protein. As you have mentioned, you are working on a membrane protein, you may be using some stabilizers to take care of the hydrophobic part of your protein. Choose the method for imidazol removal wisely (do hit and trial), as concentrating the protein on spin colums will take long time and there are chances of loss of protein because of interaction with the spin column membrane. Using desalting column may also reduce the concentration of the stabilizers or detergents in the final buffer (saturate the desalting column with the buffer [in which the protein is stable]) or try to elute the protein without imidazol (i.e. with EDTA or pH reduction). Protein science is trivial and you have to find the best possible way to get it in properly folded form, to achieve the best downstream experimental results. All the best!!
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